When intracellular calcium pools are released by agonists such as thrombin, elevation of platelet cytosolic calcium (Cai) induces tyrosine phosphorylation of a 130 Kda protein; and refilling the pools mediates dephosphorylation of this protein (Vostal et al. JBC 266:16911-16916, 1991). In the present work the 130 Kda protein was identified as vinculin by the following criteria: 1) It is detected on western blots of thrombin-activated platelets by both monoclonal anti-phosphotyrosine and anti-vinculin antibodies. 2) It associates with the platelet Triton- soluble fraction as does vinculin. 30 It reacts with monoclonal anti- vinculin when isolated by affinity chromatography on agarose linked to anti-phosphotyrosine antibody. 4) It reacts with both anti- phosphotyrosine and anti-vinculin when focused isoelectrically at a Pi of 5.4-5.8. 5) Its proteolytic fragments are reactive with both anti- phosphotyrosine and anti-vinculin. Since the level of stored calcium is known to control plasma membrane permeability to calcium and also appears to control tyrosine phosphorylation of vinculin, vinculin may be involved in regulation of platelet plasma membrane calcium channels.